Enterococcus innesii sp. nov., isolated from the wax moth Galleria mellonella

Four bacterial strains were isolated from two different colony sources of the wax moth Galleria mellonella. They were characterized by a polyphasic approach including 16S rRNA gene sequence analysis, core-genome analysis, average nucleotide identity (ANI) analysis, digital DNA–DNA hybridization (dDDH), determination of G+C content, screening of antibiotic resistance genes, and various phenotypic analyses. Initial analysis of 16S rRNA gene sequence identities indicated that strain GAL7T was potentially very closely related to Enterococcus casseliflavus and Enterococcus gallinarum , having 99.5–99.9 % sequence similarity. However, further analysis of whole genome sequences revealed a genome size of 3.69 Mb, DNA G+C content of 42.35 mol%, and low dDDH and ANI values between the genomes of strain GAL7T and closest phylogenetic relative E. casseliflavus NBRC 100478T of 59.0 and 94.5 %, respectively, indicating identification of a putative new Enterococcus species. In addition, all novel strains encoded the atypical vancomycin-resistance gene vanC-4. Results of phylogenomic, physiological and phenotypic characterization confirmed that strain GAL7T represented a novel species within the genus Enterococcus , for which the name Enterococcus innesii sp. nov. is proposed. The type strain is GAL7T (=DSM 112306T=NCTC 14608T).


INTRODUCTION
Enterococci are Gram-positive facultative anaerobes that are often diplococci, and which belong to the phylum Firmicutes, class Bacilli, order Lactobacillales and family Enterococcaceae [1,2]. They comprise a large genus of lactic acid bacteria that are tolerant to many stress conditions and can be found in a wide range of habitats including water (fresh and marine), soils, and as members of animal, human and plant microbial communities (i.e. microbiomes) [3]. From a clinical perspective, some species, such as Enterococcus faecalis and Enterococcus faecium, are associated with opportunistic infections, including bacteraemia, endocarditis and urinary tract and catheter infections [4][5][6]. Crucially, Enterococcus species have inherent resistance to many antimicrobial agents including cephaloporins and β-lactams [7,8]. They are also of further concern due to acquisition of multi-drug resistance traits, particularly rising rates of vancomycin-resistant Enterococcus strains [9], which are an increasingly common cause of infection in hospitals [10].
As highlighted above, Enterococcus species are also common animal microbiota members, and previous work has indicated that the greater wax moth, Galleria mellonella, is dominated by Enterococci [11,12], like many other species of Lepidoptera [13]. Although Galleria is a pest of honeybee (Apis mellifera) hives worldwide [14], in recent years it has gained popularity as a model host for a range of human pathogens. It has the advantages of being inexpensive, easy to use, and able to grow at 37 °C, while not being subject to the same regulations and ethical concerns as mammalian models such as mice [15][16][17]. It has also been of interest due to the ability of the larvae to metabolize polyethylene [18]. Previous research on endogenous Galleria and Enterococcus species indicates these bacteria may have a colonization-resistance function, either passively or actively, through the production of antimicrobial bacteriocins [11,19].
In this study, we isolated four bacterial strains initially identified as Enterococcus casseliflavus based on 16S rRNA gene OPEN ACCESS alignments. However, on further inspection and characterization (genomic and phenotypic) we propose a novel and putative Enterococcus species: herein named Enterococcus innesii sp. nov. These data expand our knowledge of an important model organism-associated Enterococcus species, which encodes atypical vancomycin resistance genes and is therefore also of clinical importance.

ISOLATION AND ECOLOGY
Galleria mellonella larvae were obtained from a colony grown from larvae originally sourced from Livefood UK Ltd and maintained at the John Innes Centre Entomology Facility (Norwich, UK). Galleria larvae (TruLarv) were also purchased from BioSystems Technology. Larvae were flashfrozen in liquid nitrogen, and whole guts dissected under sterile conditions (three guts were pooled into each single sample). Each sample was then homogenized in 200 µl PBS, diluted 100-fold in PBS and 50 µl spread-plated on brain heart infusion (BHI) agar plates and incubated at 37 °C for 48 h. Individual bacterial colonies were selected and grown in BHI media. Three pure isolates from distinct single colonies were taken from Galleria mellonella larvae from the John Innes Centre Entomology Facility, and one was isolated from TruLarv larvae.

GENOMIC CHARACTERIZATION
The genomes of E. innesii GAL7 T , E. innesii GAL9, E. innesii GAL10 and E. innesii TL2 were sequenced using the Nanopore MinION sequencing platform. Prior to this, FastDNA Spin Kit for Soil (MP Biomedicals) was used to extract genomic DNA from each isolate (grown up in BHI media for 48 h) following manufacturer's instructions, with an extended 3 min bead-beating procedure as described previously [20]. The sequencing library was prepared via a modified Illumina Nextera Flex low input tagmentation approach using symmetrical 24 base barcoded primers [21]. Libraries were pooled and stringently size selected on a sageELF 0.75 % cassette and fractions from 4 kb and above were pooled and put into a standard Nanopore Ligation reaction using the SQK-LSK109 kit and protocol and loaded onto a MinION following the recommended loading guidelines and run for 48 h. Basecalling was performed using Guppy version 3.6.0 (Oxford Nanopore Technologies) in high accuracy mode (model dna_r9.4.1_450bps_hac). Subsequently, high-quality pure culture genomes (genome size range: 3.6-3.8 Mb) were assembled via Unicycler version 0.4.9 [22] and further polished using Racon version 1.3.1 in the Unicycler pipeline, with a range of 13-18 in contigs and G+C content of ~42 mol% ( Table 1). Genomes were further annotated using Prokka version 1.13, with ~3800-4100 CDS predicted for these for E. innesii strains.
Initially, the 16S rRNA sequences of 61 validated Enterococcus species (60 were Enterococcus type strains) were obtained from the web server of List of Prokaryotic names with Standing in Nomenclature (LPSN; May 2021) [23,24]. Using in silico approaches, near-full-length 16S rRNA sequences (~1.5 kb) of E. innesii were extracted via bactspeciesID version 1.2 [25], Table 1. Genome statistics comparison between closely related Enterococcus species (n=10) to E. innesii strains identified by TYGS, including type strain GAL7 T [45] Previously published type strain genomes were retrieved from GenBank for analysis in this study [46]. Genome assembly statistics were extracted using sequence-stats version 0.1 [47] while genome annotation was performed using Prokka version 1.13 [48]. aligned with 16S rRNA sequences of other 61 public genomes using muscle version 3.8.31 [26], and a 16S rRNA-based maximum-likelihood phylogenetic tree was reconstructed via iq-tree version 2.0.5 with the GTR model at 1000 bootstrap replications while visualized with iTOL version 6 ( Fig. 1) [27,28]. E. innesii GAL7 T was phylogenetically positioned among E. casseliflavus, E. flavescens (re-classified as E. casseliflavus) and E. gallinarum cluster due to its 16S rRNA sequence similarity (99.53-99.93 %) [29]. However, when we compared the digital DNA-DNA hybridization (dDDH; via  Next, 10 closest-related Enterococcus strains (vs E. innesii) identified by TYGS were further examined phylogenetically at a genomic level, with antibiotic resistance genes also screened (using the resfinder database), for the four novel E. innesii strains (Fig. 2) [30]. The pangenome of these 14 strains were investigated using Roary version 3.12.0 [31] at blastp threshold at 70 % identity for inference of core genes. A total of 15 629 genes were present in this pangenome with 564 core genes and 15 065 accessory genes. Next, a core-gene alignment was generated and used to build a core-genome maximum-likelihood phylogenetic tree where it showed that E. casseliflavus NBRC100478 T was genomically distinct from E. innesii, further supported by single nucleotide polymorphism (SNP) analysis (using snp-dists version 0.7.0) that confirmed the SNP range (8-32 SNPs) among E. innesii strains (n=4) indicating strain distinction yet close genetic relatedness, while 11538-11540 SNPs were found when comparing E. innesii strains (n=4) and E. casseliflavus NBRC100478 T (Fig. 2)   GCA_901542395.1), FDAARGOS163 (GCA_001558875.2) and 4928STDY7071463 (GCA_902159265.1) are derived from human sources [37][38][39]. These isolates also demonstrated similar genome features as E. innesii sp. nov., with genome size range ~3.6-3.7 Mb and G+C ~42 mol%. These data suggest E. innesii sp. nov., may also be a clinically important species associated with novel antimicrobial resistance determinants, as vanC-4 is encoded in all these genomes, and is reported to cause opportunistic human infection.

PHENOTYPIC CHARACTERIZATION
Phenotypic characteristics were also investigated and included cell and colony morphology, motility, Gram-staining reaction, formation of endospores, oxygen relationship, growth at different temperatures, fermentation profiles of carbohydrates, catalase activity, oxidase activity, tolerance to NaCl, Voges-Proskauer reaction, urease production, pyrrolidonyl arylamidase production, hydrolysis of hippurate, deamination of arginine, pyruvate utilization, bile-aesculin tolerance test, haemolysis test, fatty acid analysis and vancomycin susceptibility testing [40]. Motility tests were carried out on E. innesii GAL7 T using motility test medium (Merck). Media were prepared according to manufacturer's instructions and outcomes were recorded after culturing for 48 h at 37 °C. The susceptibility of E. innesii GAL7 T to antibiotic vancomycin was evaluated using MIC assays on BHI agar plates (carried out in three biological replicates) as described previously [41]. Aside from motility and vancomycin susceptibility tests, all phenotypic analyses were carried out by the Identification Service, Leibniz Institute DSMZ (Germany).
E. innesii cells were coccoid-shaped, 1.0-1.5 µm long, motile and occurred in pairs or in chains under phase-contrast microscopy (Fig. 3). All E. innesii strains were Gram-positive, asporogenous, and facultatively anaerobic. Biochemical characteristics were determined using API 50CHE strips for carbohydrate utilization profiles, after incubation for up to 48 h at 37 °C (Table 2). They were capable of growth at 10-45 °C with optimum at 30-37 °C in BHI broth, with only weak growth at 45 °C, and no growth at 5 °C for up to 13 days. Growth was observed at NaCl concentrations from 0 to 8 % (w/v), with optimum growth <6.5 %. All strains were catalase-and oxidase-negative and showed no haemolytic activity. When compared to the closest related species E. casseliflavus (based on 16S rRNA analysis), E. innesii strains exhibited a distinctive metabolism in producing acid from glycerol, sorbitol, raffinose and 2-ketoglyconate, while not producing acid from turanose ( Table 2). Further phenotypic features were determined using the API rapidID32 STREP system on single strain E. innesii GAL7 T where cells were negative for urease production, hydrolysis of hippurate and pyruvate utilization (no detectable growth using sodium pyruvate as sole carbon source in mineral salt medium for 6 days at 37 °C), while positive for Voges-Proskauer reaction, pyrrolidonyl arylamidase production and arginine dihydrolase. GAL7 T cells tested positive for aesculin hydrolysis in complex medium (Bacto-Peptone, 1 g l −1 aesculin). Moreover, similar to E. gallinarum, GAL7 T cells were positive for β-glucuronidase while closest relative E. casseliflavus, and related species E. faecalis and E. faecium were all negative for this enzyme (Table 2).
Cellular fatty acids were analysed after conversion into fatty acid methyl esters (FAMEs) using a modified protocol by Miller [42]. Mixtures of the FAMEs were then separated by gas chromatography and detected by a flame ionization detector using the Sherlock Microbial Identification System (midi) based on TSBA6 database. C 14 : 0 , C 16 : 0 and C 18 : 1 ω7c were the major fatty acids in E. innesii GAL7 T . Compared to the closest phylogenetic neighbours E. casseliflavus and E. gallinarum type strains (JCM8723 T and JCM8728 T , respectively), E. innesii GAL7 T cells have a significantly higher C 14 : 0 fatty acid content at 26.12%, apparently distinctive from E. casseliflavus (7.5 %) and E. gallinarum (0.2 %) as described previously [43].
Importantly, we determined that E. innesii GAL7 T , which harboured putative atypical vancomycin resistance gene vanC-4, reduced susceptibility to vancomycin at MIC 4 µg ml −1 (vancomycin clinical breakpoint for Enterococci is >4 µg ml −1 ). This is similar to the low-level vancomycin resistance reported previously in E. casseliflavus and E. gallinarum, strains that encode the vanC resistance gene [36,44].
The type strain, GAL7 T (=DSM 112306 T =NCTC 14608 T ), was isolated from the gut of a wax moth Galleria mellonella at John Innes Centre (Norwich, UK). The genome of the type strain is characterized by a size of 3.79 Mb and a G+C content of 42.22 mol%.